Genetic variability for Russian wheat aphid, Diuraphis noxia resistance in South African wheat genotypes

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Genetic variability for Russian wheat aphid, Diuraphis noxia resistance in South African wheat genotypes

Vicki Louise Tolmay

Submitted in fulfilment of the requirements for the degree

PHILOSOPHIAE DOCTOR

in the

Faculty of Natural and Agricultural Science Department of Plant Sciences

(Plant Breeding) University of the Free State

July 2006

Promotor:

Prof C.S. van Deventer

Department of Plant Sciences, University of the Free State, Bloemfontein, South Africa

Co-promotor: Dr W.F. Tjallingii

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Contents Acknowledgements 5 List of Tables 6 List of Figures 8 Chapter 1 Introduction

Wheat as food crop in the world and South Africa ……….. 11

Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae) .….………….. 12

Biology ………...………... 12

Distribution and Biotypes ………... 13

Damage ………..………...…… 15

Chemical control measures …….……….…. 16

Non-chemical control measures ..………. 17

Plant defence and host plant resistance ……… 17

Russian wheat aphid resistance ………. 19

The study of aphid-plant interactions using EPG ……… 25

Russian wheat aphid probing behaviour ………... 27

Aims, scope and contents of this study ………. 28

References ……… 29

Chapter 2 Mechanisms of resistance and their influence on the population development of Russian wheat aphid on susceptible and resistant wheat lines under field conditions Abstract ……… 46

Introduction ………. 47

Material and methods ……… 48

Mechanisms of resistance ………. 49

D. noxia population development under field conditions: 1993-1994 seasons ………….. 50

D. noxia population development under field conditions: 2004 season .…..……….. 50

Results and Discussion ……….………... 51

Mechanisms of resistance ………. 51

D. noxia population development under field conditions: 1993-1994 seasons ……….. 54

Conclusions ……… 59

Acknowledgements ……… 60

References ………. 60

Chapter 3 Initial exploratory study of electronic monitoring of Russian wheat aphid feeding behaviour on susceptible and resistant wheat lines Abstract ……….... 63

Introduction ………...… 63

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Results ……… 71

Discussion and conclusions ………. 75

References ……… 76

Chapter 4 The influence of host plant resistance on the feeding behaviour of the Russian wheat aphid, Diuraphis noxia: Constitutive and Induced effects Abstract ………….……….. 81

Material and methods ……… 83

Results ….……… 85

Constitutive effects: Tugela vs TugelaDn [Tug-0 vs TugDn-0] ……….... 85

Induced effects: Tugela vs TugelaDn [Tug-1 vs TugDn-1; Tug-3 vs TugDn-3; Tug-5 vs TugDn-5]. 93 Main effects: Tugela vs TugelaDn [all preinfestation treatments combined] ………... 98

Discussion and Conclusions ……… 101

References ………. 104

Chapter 5 Probing behaviour of Diuraphis noxia on near-isogenic lines with resistance ex PI 137739 and PI 262660 Abstract ……….……….. 109

Material and methods ……….. 110

Results and discussion ………. 112

Conclusions ………..……….. 121

References ………..……… 122

Chapter 6 Yield retention of resistant wheat cultivars, severely infested with Russian wheat aphid, Diuraphis noxia (Kurdjumov) in South Africa Abstract ……….……….. 124

Introduction ……….……….…... 124

Material and methods ……….……….. 125

Results and discussion ………. 126

Conclusions ………….………...… 132

Acknowledgements ………... 132

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Chapter 7

General discussion and conclusions ……….… 136

References ……….. 142

Chapter 8

Summary ………. 145

Opsomming ………. 146

Declaration

I declare that the thesis hereby submitted by me for the degree Philosophiae Doctor at the University of the Free State is my own independent work and has not previously been submitted by me at another university/faculty. I furthermore cede copyright of the thesis in favour of the University of the Free State.

Vicki Tolmay 28 July 2006

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Acknowledgements

The contributions of following institutions and people are gratefully acknowledged:

The ARC-Small Grain Institute for the use of their facilities to perform the research, for the use of the data obtained and for financial support during the study.

The Agricultural Research Council and the Winter Cereal Trust who funded all the research reported in this thesis.

Prof Charl van Deventer (Department of Plant Breeding, UFS) and Dr Freddy Tjallingii (Wageningen Agricultural University, The Netherlands) for guidance and encouragement whilst supervising the research project.

Emma Mollo, Rihanlé Maré, Robbie Lindeque, Joyce Mebalo, Diederick Exley, Shadrack Mofokeng, Fanus Komen, Daniel Motaung, Esther Nhlapo, Paulina Majola, Joe Mkwanazi, Ponsto Mokoena and Alfred Mahlangu for technical support.

Marie Smith, Velimer Ninkovi , Rudolph Coetzer and Freddy Tjallingi for guidance and support with statistical analysis used in this study.

Juliette Kilian for assistance with the acquisition of literature.

The EU funded Regional Maize and Wheat Research Network for SADC (MWIRNET) for funding the purchase of EPG equipment. Dr Hugo van Niekerk, Dr George Varughese and Dr Mulugetta Mekuria for their involvement in the MWIRNET project.

A special thank you is directed to my family; exceptional people, who are very dear to me:

John without your love, encouragement and patience, not to mention the proofreading, this study would not have been possible. You were always “there” when I needed you – thank you. Sam and Nina, thanks for all the times you kept me company while I worked and for those times you managed on your own when I was busy. Mom and Dad, thanks for believing in me and teaching me to believe in myself, thanks also for all your love and support and for the many times you cooked so that I didn’t have to.

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List of Tables

Table 1.1 List of studies conducted with D. noxia resistant donor accessions ………... 23 Table 1.2 List of studies conducted with D. noxia resistant cultivars TugelaDn and Halt . 24

Table 2.1 The initial mass of D. noxia, final number, total and mean final mass of D. noxia from colony count experiment ……… 51 Table 2.2a The number of D. noxia recovered per cultivar/line for each pre-conditioning

treatment, 24 hours after infestation ……… 52

Table 2.2b The number of D. noxia recovered per cultivar/line, of the total released onto

the flats ………. 52

Table 2.3 The plant height of test entries at the onset and end of the tolerance trial ...… 53 Table 2.4 The initial and final infestation rates of the tolerance trial ……… 53 Table 2.5 Leaf area and plant dry mass of resistant lines and susceptible control in a

tolerance test ……….. 54

Table 2.6 Yield (t.ha-1), hectolitre mass (kg.hl-1) and infestation expressed as total % infested tillers and number of D. noxia per infested tiller for the 1993 season .. 54 Table 2.7 Yield (t.ha-1), hectolitre mass (kg.hl-1) and infestation expressed as total %

infested tillers and number of D. noxia per infested tiller for the 1994 season .. 55

Table 3.1 Waveform features and correlations on EPG (after Tjallingii, 1996; Prado,

1997) ………. 67

Table 3.2 EPG parameters recorded for D. noxia feeding on susceptible and resistant

South African wheat genotypes ……… 72

Table 4.1 Parameters of D. noxia probing measured by 8 hour EPG for susceptible Tugela (Tug-0) and resistant TugelaDn (TugDn-0) ……….. 87 Table 4.2 Comparison of four arbitrary classes of E1 fractions for D. noxia probing on

previously uninfested Tugela and TugelaDn ………. 90

Table 4.3 Row x Column Chi2 test of the total observed frequencies of the four classes of E1 fractions of D. noxia probing on previously uninfested Tugela and

TugelaDn ……….. 91

Table 4.4 Comparison of four arbitrary classes of E2 fractions for D. noxia probing on previously uninfested Tugela and TugelaDn ……….. 91 Table 4.5 Row x Column Chi2_{test of the total observed frequencies of the four classes}

of E2 fractions of D. noxia probing on previously uninfested Tugela and

TugelaDn ………. 91

Table 4.6 Row x Column Chi2 test of the total observed frequencies of the four classes of E2 fractions of D. noxia on Tugela and TugelaDn previously infested for

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Table 4.7 Influence of Tugela and TugelaDn on D. noxia probing measured by the non-sequential EPG parameters reflecting probing and pathway activities (all

preinfestation treatments combined) ………... 98

Table 4.8 The influence of Tugela and TugelaDn on D. noxia probing measured EPG parameters relating to phloem activities (all preinfestation treatments

combined) ………. 99

Table 4.9 Row x Column Chi2_{test of the observed frequencies of the four classes of E1}

fractions of D. noxia on Tugela and TugelaDn (all preinfestation treatments combined) ………

99 Table 4.10 Row x Column Chi2_{test of the observed frequencies of the four classes of E2}

fractions of D. noxia on Tugela and TugelaDn (all preinfestation treatments

combined) ……… 100

Table 4.11 The influence of Tugela and TugelaDn on D. noxia probing measured by sequential EPG parameters (all preinfestation treatments combined) ……….. 100 Table 5.1 EPG parameters recorded for D. noxia feeding on susceptible (Betta &

Tugela) and resistant (1684/Tugela & 2199/Tugela) South African wheat

genotypes ……… 113

Table 5.2 Row x Column Chi2_{test of the observed frequencies of E1 factions for}_D. noxia probing on Betta, Tugela, 1684/Tugela and 2199/Tugela ………. 115 Table 5.3 Row x Column Chi2 test of the observed frequencies of E2 factions for D.

noxia probing on Betta, Tugela, 1684/Tugela and 2199/Tugela ………. 117

Table 6.1 Cultivar, date of release, date of withdrawal from commercial production, resistance classification and percentage yield retained after infestation …….. 128 Table 6.2 Ranking and classification of cultivars into more resistance 9 (a) and less

resistant (b) groups (Gupta & Panchapakesan, 1979), with a 95% probability for the correct decision, for 2000, 2001 and 2003 seasons ………. 129

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List of Figures

Figure 1.1 Apterous Russian wheat aphid [Photograph:J.L. Hatting] …………..…….…… 12

Figure 2.1 Percentage tillers infested with D. noxia in the 1993 season ……… 55

Figure 2.2 Percentage tillers infested with D. noxia in the 1994 season ……… 56

Figure 2.3 Number of D. noxia per infested tiller in the 1993 season ………. 56

Figure 2.4 Number of D. noxia per infested tiller in the 1994 season ……… 57

Figure 2.5 Grain yield (t.ha-1) at Bethlehem, South Africa in the 1993 season ….………… 58

Figure 2.6 Grain yield (t.ha-1_{) at Bethlehem, South Africa in the 1994 season ……….} ₅₈

Figure 3.1 The primary circuit for EPG recording (DC system (after Tjallingii, 1996) ……… 66

Figure 3.2 Electrical penetration graph (EPG) of an aphid (after Tjallingii, 1996) ………….. 66

Figure 3.3 Close-up of leaf held in position by hairclip and EPG probe with D. noxia wired in position ………. 69

Figure 3.4 EPG set-up: GIGA-8 with eight channels, the plants and the thermograph used to record temperature within the Faraday cage ………. 69

Figure 3.5 Sum of all pathway and phloem activities (sgE1 + E12) for D. noxia probing on susceptible (Betta & Tugela) and resistant (1684/Tugela & 2199/Tugela) genotypes ……… 73

Figure 3.6 Sum of all phloem phases for D. noxia probing on susceptible (Betta & Tugela) and resistant (1684/Tugela & 2199/Tugela) genotypes ………... 73

Figure 3.7 Total number of E1 periods followed by an F or G period for D. noxia probing on susceptible (Betta & Tugela) and resistant (1684/Tugela & 2199/Tugela) genotypes ……… 74

Figure 3.8 Sequential parameters reflecting phloem access and acceptance by D. noxia ….. 75

Figure 4.1 Number of single E1 periods for D. noxia probing on susceptible Tugela and resistant TugelaDn, preinfested for none (constitutive resistance), one, three and five days (induced resistance) ………... 86

Figure 4.2 Sum of single E1 periods for D. noxia probing on susceptible Tugela and resistant TugelaDn, preinfested for none (constitutive resistance), one, three and five days (induced resistance) ……….…… 86

Figure 4.3 Mean duration of E2 fractions for D. noxia probing on susceptible Tugela and resistant TugelaDn, preinfested for none (constitutive resistance), one, three and five days (induced resistance) ………. 89

Figure 4.4 Sum of E2 fractions for D. noxia probing on susceptible Tugela and resistant TugelaDn, preinfested for none (constitutive resistance), one, three and five days (induced resistance) ……….. 89

Figure 4.5 Number of F and G periods for D. noxia probing on susceptible Tugela and resistant TugelaDn, preinfested for none (constitutive resistance), one, three and five days (induced resistance) ………. 92

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Figure 4.6 Sum of F and G periods for D. noxia probing on susceptible Tugela and resistant TugelaDn, preinfested for none (constitutive resistance), one, three

and five days (induced resistance) ……….. 92

Figure 4.7 Number of path periods for D. noxia probing on susceptible Tugela and resistant TugelaDn, preinfested for one, three and five days (induced resistance) …………

93 Figure 4.8 Sum of phloem for D. noxia probing on susceptible Tugela and resistant

TugelaDn, preinfested for one, three and five days (induced resistance) …….... 94 Figure 4.9 Mean duration of single E1 periods for D. noxia probing on susceptible Tugela

and resistant TugelaDn, preinfested for one, three and five days (induced

resistance) ………... 94

Figure 4.10 Mean duration of E12 periods for D. noxia probing on susceptible Tugela and resistant TugelaDn, preinfested for one, three and five days (induced resistance) .

95 Figure 4.11 Sum of E12 periods for D. noxia probing on susceptible Tugela and resistant

TugelaDn, preinfested for one, three and five days (induced resistance) ……… 95 Figure 4.12 Time to 1st sustained E2 (>10 min) in experiment for D. noxia probing on

susceptible Tugela and resistant TugelaDn, preinfested for one, three and five

days (induced resistance) ………. 97

Figure 4.13 Cluster analyses to illustrate the relative association between probing behaviour of D. noxia on resistant and susceptible genotypes pre-conditioned

for none, one, three and five days ………. 101

Figure 5.1 Duration of first probe for D. noxia probing on Betta, Tugela, 1684/Tugela and

2199/Tugela ………..……. 112

Figure 5.2 Number of probes shorter than 3 minutes before the first phloem period for D. noxia probing on Betta, Tugela, 1684/Tugela and 2199/Tugela ……… 114 Figure 5.3 Sum of single E1 periods and E1 fractions for D. noxia probing on Betta,

Tugela, 1684/Tugela and 2199/Tugela ………. 114

Figure 5.4 Mean duration of E12 periods, E1 and E2 fractions for D. noxia probing on Betta, Tugela, 1684/Tugela and 2199/Tugela ……….. 116 Figure 5.5 Maximum duration of E12 periods, E1 and E2 fractions for D. noxia probing on

Betta, Tugela, 1684/Tugela and 2199/Tugela ………... 117 Figure 5.6 Time to the first sustained phloem ingestion for D. noxia probing on Betta,

Tugela, 1684/Tugela and 2199/Tugela ……….… 118

Figure 5.7 Percentage time in E2 after first sustained phloem ingestion for D. noxia

probing on Betta, Tugela, 1684/Tugela and 2199/Tugela ……….…… 118 Figure 5.8 Number of probes after the first sustained phloem ingestion for D. noxia probing

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Figure 6.1 Long term average pre-season (December to June) and in-season (July to November) rainfall (mm) as well as the rainfall (mm) measured at Bethlehem

during 2000, 2001 and 2003 ……… 127

Figure 6.2 AMMI biplot of the first Interaction Principle Component Analysis axes for percentage yield retained after severe Russian wheat aphid infestation. Grand mean % yield retained for the trial is 82.01 ……… 131

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Chapter 1 Introduction

Wheat as food crop in the world and South Africa.

Ninety-five per cent of the world’s calories now come from only 30 crops, and 50% from just four: rice, maize, wheat and potato (Webb, 2000). Wheat is the most widely grown cereal crop in the world, and the world trade for wheat is greater than for all other crops combined (Curtis, 2002). Wheat originated in the Fertile Crescent of the Middle East from where it spread to North Africa, Eurasia, Western Europe, the Americas and the Southern hemisphere (Pagesse, 2000). Feldman (2000) describes the origin of cultivated wheats, which are divided into three main groups: diploids [2n=2x=14] (einkorn), tetraploids [2n=4x=28] (emmer, durum, rivet, Polish and Persian wheat) and hexaploids [2n=6x=42] (spelt, bread, club and Indian shot wheat). Hexaploid bread wheat, Triticum aestivum, presumably originated in northwestern Iran or northeastern Turkey as a result of a hybridisation between tetraploid wheat and diploid Aegilops tauschii some >8000 years BC. Due to the polyploid genetic structure of Triticum species and the associated genetic diversity, these plants have successfully been established throughout the world in varying environments.

The average global world wheat production from 1995-1999 was 584 million tons per annum (Maratheé and Gómez-MacPherson, 2000) and world production is expected to reach 860 million tons per annum by 2030. For the period 1995-1997 the average wheat consumption in the world was 73kg/person/year compared to that for sub-Saharan Africa in the same period of only 15kg/person/year. The per capita wheat consumption in South Africa is closer to the world average at 75.6kg/person/year (Payne, Wanjama and Girma, 2000). The annual wheat production in South Africa ranges from 1.7–2.7 million tons per annum (NDA, 2000) depending on the season and with an annual consumption of 2.8 million tons per annum South Africa is a net importer of wheat. Profit margins for producers are slim, with prices determined in a free market environment, linked to the international trade and influenced by the Rand/USD exchange rate.

Wheat is cultivated in three distinct production areas in South Africa. The mediterranean, winter rainfall region in the Western Cape grows dryland spring wheat and contributes approximately 30% of the annual yield while 20% of the annual yield is produced by irrigated spring wheat grown in the central irrigation areas including the Northern Cape. The remaining 50% of annual production is derived from dryland winter and intermediate/facultative wheat grown in the summer rainfall region on stored soil moisture that was accumulated during the preceding summer and autumn. This is a unique production system characterised by low seeding rates of 15-30 kg/ha using cultivars with long coleoptiles (>6cm) and a high tillering ability. Wheat is planted from May to August and harvested from late November to January depending on the season. The summer rainfall season stretches from October to February, but earlier spring rains can occur from August. Abiotic stress factors in this system include aluminium toxicity due to acid soils and pre-harvest sprouting due to rainfall during the harvest season while biotic stress factors include both diseases such as stripe

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rust (Puccinia Westend f. sp. striiformis Eriks.), leaf rust (Puccinia triticina Eriks.), take-all (Gaeumannomyces graminis var. tritici), glume blotch (Septoria nodorum Berk.) and crown rot (Fusarium spp) as well as a number of insect pests of which Russian wheat aphid, Diuraphis noxia

(Kurdjumov), is the most important.

Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae)

D. noxia is a small (<2.0 mm), spindle shaped, pale yellow-green to grey-green aphid with extremely short antennae (Figure 1.1). The dorsal process of the 8th abdominal tergite gives the impression of a "double tail" when viewed laterally and the siphunculi are not prominent (Du Toit and Aalbersberg, 1980; Walters, Penn, Du Toit, Botha, Aalbersberg, Hewitt and Broodryk, 1980). Reviews and bibliographies of the Russian wheat aphid have been published by Hughes (1988), Kovalev, Poprawski, Stekolshchikov, Vereshchagina and Gandrabur (1991) and Poprawski, Underwood, Mercadier and Gruber (1992).

FIGURE 1.1: Apterous Russian wheat aphid [Photograph:J.L. Hatting]

Biology

D. noxia has four nymphal instars and an adult stage. A simple key for the diagnosis of the instars, using morphology of the antennae, caudae and wing buds in conjunction with ratios between antennal segment lengths, was developed by Aalbersberg, Van der Westhuizen and Hewitt (1987). As far as is known, only female D. noxia occur in South Africa and reproduction is parthenogenetic with both the alate and apterous forms of D. noxia being reproductive. Alate formation occurs when the host plant is under stress or when the host plant no longer provides a favourable habitat (Walters et al., 1980). D. noxia is able to survive temperatures as low as –20 °C (Butts, 1992). Nymph production is optimal at temperatures higher than 5 °C and lower than 20 °C and can peak at 4 nymphs per day with a total of 70 nymphs produced per female in a typical lifetime (Robinson, 1992).

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D. noxia is known to reproduce sexually in other parts of the world. Kiriac, Gruber, Poprawski, Halbert and Elberson (1990) reported on the occurrence of sexual morphs (both oviparae and males) of Russian wheat aphid in several locations in the Soviet Union but only the presence of oviparae in Idaho and Oregon in the USA, speculating that North American D. noxia my be gynocyclic. Basky (1993b) reported D. noxia in Hungary to be holocyclic. Clúa, Castro, Ramos, Giménez, Vasicek, Chidichimo and Dixon (2004) reported that only 20% of the 22 D. noxia clones collected throughout Argentina and Chile produced sexuals irrespective of the host they were collected from, the period of the year, region, current host, day length and average temperature of the rearing conditions.

Distribution and Biotypes

D. noxia is endemic to central Asia, southern Russia, countries bordering the Mediterranean Sea, Iran and Afghanistan (Durr, 1983; Hewitt, Van Niekerk, Walters, Kriel and Fouchè, 1984; Dolatii, Ghareyazie, Moharramipour and Noori-Daloii, 2005) but now occurs in virtually all the major small grain production regions of the world except northeastern China (Robinson, 1992) and Australia (Hughes and Maywald, 1990) where it is listed in the Grainguard Threat Data Sheet for the wheat Industry (Botha and Hardie, 2000).

The earliest published reference to D. noxia as a pest was in the Crimea (Mokrzhetsky, 1901 as quoted by Kovalev et al., 1991). Sporadic outbreaks of this pest have occurred in the former USSR since, with losses of 75% reported due to infestations of this aphid in 1912 (Mokrzhetski (1914) as cited by Halbert and Stoetzel, 1998). More recently damage caused by D. noxia was restricted to the steppe zone of the Ukraine and Russian Soviet Federated Socialist Republic (Voronin, Shapiro and Pukinskaya, 1988 as quoted by Kovalev et al., 1991). An epidemic was reported in 1962 in the Konya Province in Turkey where crop losses of 25-50% occurred (Elmali, 1998). In Africa D. noxia

was reported in the Wukro (Atsbi) and Adigrat regions of Ethiopia in 1972/73 and from the western Welo region in 1974. By 1976 D. noxia was widespread in all the barley and wheat growing areas of Ethiopia (Haile, 1981) and was considered to be the leading pest of cereals in the highlands of Ethiopia. Barley grain yield losses of 41-71% were reported in Ethiopia by Miller and Adugna (1998). In South Africa, D. noxia was first reported as a pest of wheat in 1978 (Walters, 1984) and has occurred annually since. Attia and El-Kady (1988) observed D. noxia on wheat and barley in Beni-Suef Province in Egypt during 1985, noting subsequent spread to other cereal producing areas of Egypt. In 1995 D. noxia was also reported in Kenya where yield losses of 25-90% occurred (Kiplagat, 2005). In 1980 this pest was found to be present in Mexico (Gilchrist, Rodriguez and Burnett, 1984), the first report of its presence on the American continents. By 1986 it was reported to be present in Texas and is now found in 17 western states of the United States (Miller, Porter, Burd, Mornhinweg and Burton, 1994). In July 1988, D. noxia was detected in Canada. It was first recorded in southern Alberta and spread to Saskatchewan and British Columbia by the end of the year (Jones, Byers, Butts and Harris, 1989). Russian wheat aphid has been reported in Chile and Argentina where it was initially reported in 1988 and 1992 respectively (Ortego and Delfino, 1994 as cited by Clúa et al., 2004). It was found in the main cereal-producing region of Argentina in 1994 (Bellone and Amaraz,

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1995 as cited by Clúa et al., 2004) and then spread northwards and eastwards infesting T. aestivum and T. durum in 1995 (Castro, Ramos, Vasicek, Worland, Giménez, Clúa and Suárez, 2001). In Europe, D. noxia is also known to occur in Hungary (Basky, 1993b) although it is not an economically significant pest there (Basky, 1993a; Tolmay, Basky and Lang, 2001), Serbia (Petrovi , 1992 as cited byStarý, Basky, Tanigoshi and Tomanovic , 2005), Slovakia (Lukáš, Toth, Vráblová, Lukášová and Cagán, 1999 as cited by Starý et al., 2005), Croatia (Bar i and uljak, 2002 as cited by Starý et al., 2005), Romania (Holman and Pintera, 1981 as cited by Starý et al., 2005) and Austria (Cate, 2000 as cited by Starý et al., 2005). Zhang (1991) as cited by Botha and Hardie (2000) reported that D. noxia had been known to occur in the Xinjiang-Uiger Autonomous Region of the Peoples Republic of China for decades, but that it had not spread to the major wheat growing areas of central China.

Several studies have indicated the presence of diversity in D. noxia populations found in various parts of the world. Puterka, Burd and Burton (1992) have shown that D. noxia from different parts of the world vary in their reaction to resistant wheat lines in the USA. Puterka, Black, Steiner and Burton (1993)found strong similarities between United States populations of D. noxia and collections from South Africa, Mexico, France and Turkey with most variation detected among populations from the Middle East and southern Russia. Differences have been reported between D. noxia in South Africa and Syria (Black, DuTeau, Puterka, Nechols and Pettorini, 1992), as well as between South Africa and Hungary (Basky, Hopper, Jordaan and Saayman, 2001). Black et al. (1992) amplified DNA from individual D. noxia nymphs and adults collected from South Africa, and found that there appeared to be two genotypic patterns in the South African Russian wheat aphid population while the Syrian population appeared homogenous.

In the USA, a study by Shufran, Burd and Webster (1997) reported baseline information on the biotipic status of D. noxia prior to the commercial planting of resistant cultivars indicating no genotypic variation in aphid clones collected from various localities on barley and wheat. The detection of a new biotype of D. noxia in Colorado in 2003, which is virulent to commercially resistant cultivars containing the Dn4 resistance gene (Haley, Peairs, Walker, Rudolph and Randolph, 2004) sparked renewed interest in studying biotypes of D. noxia throughout the world. Belay, Smith and Stauffer (2004) reported finding no biotypic variation within Ethiopian D. noxia based on damage ratings of various resistant lines, however the Ethiopian, Czech and Chilean biotypes of D. noxia were all virulent to Dn4

(Smith, Belay, Stauffer, Starý, Kubeckova and Starkey, 2004). The genetic marker study linked to this work was not successful in detecting significant variation in polymorphisms to detect biotypic variation. Dolatti et al. (2005) studied the regional diversity and host adaptaion of Iranian D. noxia

populations finding that one or a few widespread genotypes occurred along with many rare genotypes. Differentiation was also observed between D. noxia collected off barley and wheat. D. noxia is native to Iran and the high genetic diversity reported by this study can be explained by the possibility of sexual reproduction of the aphid in this region as well as the long period of time that the aphid has been present in the area. The presence of a resistance breaking bioype of D. noxia

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cultivars marketed as resistant during the 2005 season are severely damaged by the new biotype, which has not yet been characterised against the international differential set of resistance genes.

Damage

Yield losses due to D. noxia are severe with individual plant losses as high as 90% possible (Du Toit and Walters, 1984). Robinson (1992) recorded crop losses of 68% in Ethiopia and 35-60% in South Africa for wheat. Yield losses in Ethiopia for barley were estimated to be between 41-71%. In 1993 the yield losses caused by D. noxia in South Africa amounted to approximately R30 million (Swart, 1999) with approximately R15 million spent on chemical control annually (Cilliers, Tolmay and Van Niekerk, 1992). In the United States losses due to D. noxia have been quantified as running into millions of dollars annually. The cumulative economic loss (1987-1993) attributed to D. noxia in the United States exceeds $890 million, with approximately $83 million being spent on control, $349 million in lost production and $460 million in additional lost economic activity in local communities (Webster and Amosson, 1994).

The symptoms of D. noxia infestation are very distinct. Typical white, yellow and purple to reddish-purple longitudinal streaks occur on the leaves of plants infested with D. noxia. The aphids are found mainly on the adaxial surface of the newest growth, in the axils of leaves or within rolled leaves. Heavy infestations in young plants cause the tillers to become prostrate, while heavy infestations in later growth stages cause the ears to become trapped in the rolled flag leaf (Walters et al., 1980). Severe damage is associated with these symptoms. The toxin or biochemical reaction that causes the damage has not yet been identified, though the effects are well known. D. noxia infestation leads to a drastic reduction in chlorophyll content (Kruger and Hewitt, 1984) and reduced photosynthetic ability (Fouché, Verhoeven, Hewitt, Walters, Kriel and de Jager, 1984) which, when combined with the characteristic leaf rolling that occurs, causes a considerable loss of effective leaf area of susceptible plants (Walters et al., 1980). Matsiliza (2003) showed that D. noxia feed preferentially from thin-walled sieve tubes in sink as well as source leaves of wheat and that the small longitudinal bundles were preferred. Eighty three percent of stylet tracks in sink leaf material terminated in thin-walled sieve tubes while on source leaf tissue 95% of stylet tracks also terminated in thin-walled sieve tubes. It was postulated that the preference for these veins is likely to be related to the quality and quantity of assimilates in them as these veins have been implicated in the assimilate loading in source leaves. By feeding on minor rather than major veins the aphid has the advantage of a shorter pathway to the sieve tubes, less sclerenchyma to impede the passage of the stylets and a food source which may be richer in both sugars and proteins as the smaller veins have been implicated in the loading and unloading of assimilates. Using analine blue stain Botha and Matsiliza (2004) reported that D. noxia infested leaf tissue (wheat cv Adamtas) was heavily callosed, with callose deposited between the plasma membrane and the cell wall, not only within the phloem tissue, but also in neighbouring vascular parenchymea cells. Deposition of wound callose was found to have disrupted phloem transport and thus the export of photo-assimilate from

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the leaves. Matsiliza (2003) confirmed that typical of most aphids, D. noxia probes the leaf between epidermal cells or through the stomata and proceeds on an intercellular pathway through the mesophyll cells to the vascular tissue from where pathway is intracellular, near and inside the bundle and that feeding aphids form local sinks, once their stylets have penetrated the functional phloem. Burd and Burton (1992) showed that D. noxia infestation resulted in water imbalances in the host plant, expressed as a loss of turgor and reduced growth. Substantial reductions in plant biomass also occur (Burd and Burton, 1992).

Many factors influence D. noxia damage. It is widely accepted that D. noxia show a preference for stressed host plants and that plants grown under drought stress or low nitrogen levels are more damaged. Johnson, Ni, McLendon, Jacobsen and Wraith (1998) reported that drought stressed wheat plants infested with D. noxia showed higher leaf surface temperatures and argued that D. noxia inside a longitudinally rolled leaf could maintain higher body temperatures and may thus attain maximal developmental and reproductive rates. The level of infestation, the growth stage of the host plant and the duration of the infestation all influence the severity of the damage caused by D. noxia. Du Toit and Walters (1984) concluded that wheat plants were most sensitive to D. noxia

infestation from the flag leaf stage to flower initiation. Burd and Burton (1992) indicated that the duration of infestation, rather than the level of infestation may be more important when damage is caused to the host plant. In colder climates reduced coldhardiness and therefore also plant survival and yield due to D. noxia infestation were reported by Storlie, Talbert, Taylor, Ferguson and Brown (1993). This was found to be associated with higher osmotic potentials and lower fructan content in winter wheat infested with D. noxia.

Phloem feeding insects are well known for their ability to transmit plant viruses. D. noxia was reported as a vector of barley yellow dwarf virus, brome mosaic virus and barley stripe mosaic virus (Von Wechmar, 1984). Cronjè (1990) found that D. noxia in South Africa, was not an effective vector of brome mosaic virus, with only 20 percent successful transmission under controlled conditions. Researchers in the United States have been unable to confirm any significant transmission of viruses by D. noxia (Damsteegt, Gildow, Hewings and Carroll, 1992; Halbert, Connelly, Bishop and Blackmer, 1992).

Chemical control measures

The effective control of Russian wheat aphid has been a significant challenge facing wheat producers and researchers alike throughout the regions where this pest occurs. Due to the aphids’ habit of feeding within the rolled leaf whorl, options for chemical control of D. noxia have been limited to the use of systemic insecticides such as disulfoton, dimethoate and demeton-s-methyl, vapour action insecticides such as chlorpyriphos and parathion which can penetrate the rolled leaf and more recently seed dressings such as imidacloprid and thiametoxam (Nel, Crause and Khelawanlall, 2002). In many countries the use of some of the aforementioned insecticides was

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discontinued due to environmental and safety concerns leaving very few chemical control options available to producers.

Non-chemical control measures

In South Africa damage to wheat crops can be limited by the use of systemic insecticides, but the large-scale use of insecticides has been discontinued as farmers are now planting resistant cultivars to control this pest (Tolmay, Prinsloo and Hatting, 2000) as a key component of an integrated control strategy against D. noxia in both commercial and small-scale production situations. World-wide the use of insect-resistant cultivars is seen as one of the most desirable alternatives to insecticides because of their low cost and environmentally friendly action (Burton, Porter, Baker, Webster, Burd and Puterka, 1991; Quisenberry and Schotzko, 1994). Resistance breeding against D. noxia takes place in South Africa (Tolmay and Van Deventer, 2005; Van Niekerk, 2001), the USA (Quick, Ellis, Normann, Stromberger, Shanahan, Peairs, Rudolph, and Lorenz, 1996), at CIMMYT in Mexico and ICARDA in Syria (Robinson, 1992) and pre-emptively in Australia (Botha and Hardie, 2000).

Plant defence and host plant resistance

Host plant resistance to insect pests of crop plants is generally seen as an effective, environmentally responsible, economically and socially acceptable method of pest control which plays an integral role in sustainable agricultural systems (Wiseman, 1999). Pest resistant crops offer a solution that can be tailored to meet the specific need of producers while usually offering more benefits than drawbacks for the environment. The most important benefit of a pest resistant crop is the fact that the pest control occurs independently of the managerial ability, skill and resource level of the producer (Tolmay, 2001). Host plant resistance has been used as a control measure for various agricultural pests for many years (Smith, 1989).

Painter (1951) explained host plant resistance by using three functional categories, namely antibiosis, non-preference (antixenosis) and tolerance which describe the pest-host interaction. Antibiosis describes the negative influence of the plant on the biology of an insect attempting to use that plant as host (Smith, 1989). This may be expressed as reduced body size and mass, prolonged periods of development in the immature stages, reduced fecundity or failure to pupate or eclose. Antixenosis, the inability of a plant to serve as a host, is caused by physical or chemical plant factors that repel or deter insects from feeding or oviposition (Smith, 1989). Tolerance indicates the plant's ability to withstand or compensate for insect damage (Smith, 1989). Known components of this form of resistance include general vigour, compensatory growth, wound healing, mechanical support in tissues and organs and changes in photosynthetic partitioning. Environmental factors, however may affect tolerance more than other types of resistance (Pedigo, 1989). The mechanism of resistance in a specific line will influence the efficacy of the line in

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controlling field populations of the pest and may in part determine the longevity of the resistance under field conditions by influencing the formation of a biotype (Gallun, 1972). Many factors play a role in the expression of host plant resistance and its effect on the target pest when deployed in resistant cultivars in the field.

The actual nature of the resistance within the plant itself has also been studied extensively (Agrawal, Tuzen and Bent, 2000). Gatehouse (2002) defines constitutive resistance as those morphological and chemical factors that are present in a plant prior to attack also known as passive defence, in contrast to induced resistance, which is defined as an active response by the plant to attack. Morphological factors include general tissue toughness, silica, calcium carbonate or lignin surrounding vascular bundles, leaf hairs and epicuticular wax, which form physical barriers to attack. Chemical factors are plant products that have some antimicrobial or antiherbivore (deter, poison, starve) activity and are often due to phenolics, alkaloids and proteins (Van der Westhuizen, 2004). Most protein-based defences known to date have an anti-nutritive effect on herbivores, destroying or preventing the assimilation of nutrients by the insect, thereby slowing its growth and development (Constabel, 2000). Overall, in terms of chemical factors, there is often no inherent difference between the chemistry of constitutive and induced defences with the accumulation / up-regulation pre-existing compounds being induced by herbivore damage.

It is generally accepted that the expression of constitutive resistance in plants is associated with a fitness cost that accrues when pests are absent and the magnitude of these costs is thought to explain why susceptible genotypes persist in plant populations (Cipollini, Purrington and Bergelson, 2003). Induced defences are only produced by plants under attack from pests. This defence can be localised, or systemic. Induced responses in plants to herbivore attack are thought to be a form of adaptive phenotypic plasticity, saving metabolic costs by expressing defences only when necessary (Cipollini et al., 2003). The costs of induced resistance responses have been shown to accrue as a result of the allocation of resources towards defence production and away from primary metabolism or even as a result of auto toxicity of defence chemicals. Ecological costs of induced resistance may include increased susceptibility to untargeted herbivores as shown by Agrawal, Gorski and Tallamy (1999) where increased levels of cucurbitacins in cucumber plants provided resistance to generalist arthropod herbivores while acting as a feeding stimulant for specialist beetles. In radishes there is evidence that increased resistance to herbivores may reduce the attractiveness of plants to pollinators (Karban and Nagasaka, 2004). The basic response of plants to herbivory is the wounding response, which is both local and systemic and usually involves multiple signalling pathways. Insects that feed on the content of the vascular tissue and avoid extensive tissue damage evade the wounding response and have been reported to activate the same defence response as pathogens (Gatehouse, 2002; Kaloshian and Walling, 2005).

In most cases of defences induced by insect herbivory, for both the wounding and pathogenesis pathways, saliva plays an important role in the elicitation of plant defense responses (Felton and

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Eichenseer, 2000). Insect saliva performs a multitude of functions amongst others digestion, lubrication of the mouthparts, pH regulation and in some cases suppression of host response. Aphids secrete two types of saliva (Miles, 1959); one that gels soon after secretion, forming a sheath around the stylets and the other a ‘watery saliva’ which is secreted during ingestion. The stylet sheath is though to assist aphids by holding the stylets in place while probing, sealing wounds and fluid loss when individual cells are punctured, preventing ingestion of unacceptable fluids, preventing signals produced by aphid feeding from diffusing out of the wound area and adsorbing antifeedant phenolics (Felton and Eichenseer, 2000). A comprehensive paper on the saliva of Hemiptera was published by Miles (1972).

In order to effectively utilise plant defense mechanisms against pests in a breeding programme a comprehensive understanding of the mechanisms that underlie resistance responses is needed (Van der Westhuizen, 2004). Despite the many advantages of exploiting naturally occurring plant defences there are numerous challenges associated with this practice; combining resistance with high yield and good quality being one of the most important (Prado, 1997; Tolmay, 2001; Van der Westhuizen, 2004). Plant defence and the expression of resistance are affected by various factors including nutrient availability (Glynn, Herms, Egawa, Hansen and Mattson, 2003) and general physiological condition of the plant. Furthermore, though effective, resistance may not necessarily limit pest outbreaks. Morris and Dwyer (1997) have shown that constitutive resistance influences the speed of a herbivore invasion by influencing the spatial dynamics of herbivore populations most while both constitutive and inducible resistance alter demographically important rates of herbivore birth, growth and survival. Furthermore, it was demonstrated that if levels of constitutive resistance are high and herbivore movement is sensitive to host quality, the rate of herbivore spread could in effect be accelerated even though the intrinsic rate of increase is reduced.

Russian wheat aphid resistance

Since the first report of host plant resistance to D. noxia in Triticum monococcum (Einkorn), line A 544, and other T. monococcum/T. durum amphiploids (Du Toit and Van Niekerk, 1985) many other sources of resistance have been described. Though not exhaustive as others have been reported since then, the most comprehensive review of D. noxia resistance sources was compiled by Souza (1998) who listed 98 accessions of Triticum aestivum and related species reported resistant to D. noxia by various authors. Besides that reported in bread wheat, D. noxia resistance has been reported in T. monococcum, T. turgidum, T. dicoccum, Secale cereale, X Tritcosecale, T. tauchii,

Hordeum vulgare, H. bulbosum, H. bogdani and H. brevisubulatum. Ten resistance genes have been identified and their chromosome location determined. Most D. noxia resistance genes identified to date are either located to the D chromosomes or to the rye translocation of wheat (Lage, Skovmand and Andersen, 2004). Seven of these genes namelyDn1 (Marais and Du Toit, 1993; Schroeder-Teeter, Zemetra, Schotzko, Smith and Rafi, 1994), Dn2 (Ma, Saidi, Quick and Lapitan, 1998), Dn5 (Marais and du Toit, 1993), Dn6 (Lui, Smith and Gill, 2002), as well as Dn8,

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Dn9 and Dnx (Lui, Smith, Gill and Tolmay, 2001) have been located on the 7D chromosome of wheat. Dn4 (Ma et al., 1998) was located to the 1D chromosome of wheat while dn3 was found in a diploid D-genome Aegilops tauchii line. Marais, Wessels, Horn and Du Toit (1998) reported Dn7

on a 1BL/1RS translocation from rye, Secale cereale. Efforts to broaden the genetic base for resistance have been attempted with D. noxia resistant intergeneric hybrids developed by Aung (1991) from crosses between Hordeum vulgare and Elymus trachycaulus and more recently with resistant synthetic hexaploid wheat developed from interspecific crosses of Triticum dicoccum and

Aegilops tauchii where the resistance gene(s) have been shown to be located on the A and/or B genomes, therefore presumed different to previously identified resistance genes (Lage et al., 2004). D. noxia resistance identified in bread wheat has, however been the most deployed in breeding programmes to date.

In South Africa, the use of D. noxia resistant cultivars was made possible through the discovery of host plant resistance against this pest, in bread wheat, by Du Toit (1987; 1988; 1992). The first crosses between the resistance donors and adapted South African bread wheat cultivars were made in mid 1986, the first field evaluations of back-cross progeny were undertaken in 1989 (Du Toit, 1993) and the first cultivar, Tugela-Dn, released in 1992 (Van Niekerk, 2001). D. noxia

resistant cultivars released for commercial use in South Africa have been shown to have a yield advantage above susceptible cultivars in farmers fields (Marasas, Anandajayasekeram, Tolmay, Martella, Purchase and Prinsloo, 1997). The adoption of D. noxia resistant cultivars in South Africa as documented by Marasas et al. (1997) was found to be limited only by the availability of seed. These cultivars provided a welcome alternative as the cost of systemic insecticides became prohibitive especially where harsh climatic conditions reduced their efficacy (Du Toit, 1988; 1992). It was estimated that between 70 and 85% of the area planted to wheat in 2001 was under resistant cultivars. These cultivars saved wheat producers approximately ZAR 120.00 (one tenth of the income per ton) per hectare by eliminating the need for chemical control making it easier to produce wheat at the same price it would cost to import wheat bought on the global market (Tolmay, 2001). The benefit was noticed in the environment as well; all insecticides, except two namely imidacloprid and amethoxam, registered for the control of Russian wheat aphid in South Africa were broad-spectrum systemic or contact organophosphates (LD50 2-70mg/kg). Due to the

rapid adoption of resistant cultivars the average area treated with insecticides decreased from 85% in 1990 to 30% in 1997 (Marasas, 1999). By 2006 a total of 27 cultivars with D. noxia resistance had been released in South Africa namely Betta-Dn, Caledon, Elands, Gariep, Komati, Limpopo, Matlabas, Nossob, PAN 3235, PAN 3364, PAN 3144, SST 124, SST 322, SST 333, SST 334, SST 347, SST 363, SST 367, SST 399, SST 935, SST 936, SST 946, SST 966, SST 972, SST 983, Tarka and Tugela-Dn (Dr A. Barnard, personal communication)1.

The international trend for reducing the impact of D. noxia on small grains is the use of Russian wheat aphid resistant cultivars (Webster, Starks and Burton, 1987; Du Toit, 1989b; Robinson,

1 Dr Annelie Barnard, Programme Manager: Crop Science, ARC-Small Grain Institute, Private Bag x29, Bethlehem, 9700. E-mail: BarnardA@arc.agric.za

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Delgado, Vivar and Burnett, 1992). To date resistant cultivars have been developed and released not only in South Africa (Tolmay and Van Deventer, 2005) but in the USA in Colorado (Randolph, Peairs, Koch, Walker, Stubbs, Quick and Haley, 2005) and Kansas (Qureshi, Jyoti and Michaud, 2005). This proved to be a worthwhile investment with researchers in the USA estimating the return on investment in developing the resistant cultivar Halt to be 13:1 (Webster and Kenkel, 1999) while Marasas et al. (1997) reported a Rate Of Return benefit to society from the yield gains of resistant cultivars in South Africa at 34%.

Du Toit (1989a) reported the resistance in PI 137739 and PI 260660 to D. noxia to be governed by single dominant genes, which probably differ from each other. These genes were designated Dn1 and Dn2 respectively and the mechanisms of resistance in the donor lines shown to be antibiosis and antixenosis (Du Toit, 1987, 1989b). Various other authors also studied these original sources of D. noxia resistance, PI 137739 and PI 262660. Smith, Schotzko, Zemetra and Souza (1992) evaluated these lines under greenhouse conditions in Idaho to determine the categories of resistance. Based on percentage reduction in plant height it was concluded that both lines possessed a significant level of tolerance to D. noxia feeding. D. noxia maintained on these lines displayed reduced reproductive rates 21 days after infestation, indicating the presence of low-level antibiosis. D. noxia on PI 137739 was found to have a significantly lower reproduction rate than on PI 262660 and Stephens, the susceptible control, in a trial conducted by Quisenberry and Schotzko (1994). This indicated that PI 137739 showed antibiosis in contrast to PI 262660 which had higher plant growth, dry weight and moisture while expressing higher leaf chlorosis and mid-leaf rolling indicating tolerance. Mowry (1994) found that antibiosis in the lines PI 137739 and PI 262660 could not be detected statistically when uninfested plants were compared to susceptible controls, but that D. noxia performance was significantly less on these lines when Barley Yellow Dwarf Virus (BYDV) - infested plants were compared, indicating that plant stress influences the expression of

D. noxia antibiosis.

A large number of studies have been conducted on various aspects of the biochemical and physical characteristics of D. noxia resistance. Both donor accessions and improved lines have been studied. Studies conducted on donor accessions are listed in Table 1.1. Regarding improved lines, various authors have studied two D. noxia resistant cultivars namely TugelaDn and Halt, developed in South Africa and Colorado (USA) respectively and their near-isogenic counterparts. A substantial amount of information is known regarding the nature and effect of the resistance in these two genotypes. TugelaDn [PI 591932] with resistance ex PI 137739 (Van Niekerk, 2001; Tolmay, Du Toit and Smith, 2006) and Halt [PI 584505] with resistance ex PI 372129 (Quick et al.,

1996), are accepted as containing the D. noxia resistance genes Dn1 and Dn4 respectively. The studies conducted using these lines have been listed in Table 1.2.

Available information suggests that D. noxia resistance includes both constitutive and induced elements with induced responses being the more important component. Examination of trichome presence and leaf epicuticular wax ultrastructure by Bahlmann, Govender and Botha (2003)

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showed that susceptible Tugela had 1.7 times fewer trichomes per mm2 (=16.39) than resistant Tugela-Dn (=28), while there was no difference in trichome length and epicuticular wax ultrastructure between the cultivars. Ni and Quisenberry (1997b) also reported a high density (=21.33 trichomes.mm-2_{) of short trichomes (68.1 µm) on the adaxial leaf surface of the resistant}

cultivar Halt. Furthermore, a study on the distribution of D. noxia salivary sheaths on resistant Halt and susceptible Arapahoe wheat by Ni and Quisenberry (1997a) showed that the majority of sheaths were made by intercellular penetration of leaf epidermal cells on both cultivars but significantly more sheaths were made through leaf stomata on Halt than on Arapahoe possibly eluding to a need for easier access on the resistant host. Most sheaths terminated in the vascular bundles on both cultivars, with no significant difference being recorded between the cultivars. In terms of induced defences, studies conducted regarding the biochemistry of D. noxia resistance indicate that the response is not a wounding response usually characteristic of herbivore damage, but a typical hypersensitive response (HR) more characteristic of pathogenesis (Van der Westhuizen, 2005). D. noxia infestation induced enhanced expression/synthesis of two polypeptides (100 kD [nuclear encoded] and 56 kD [organel encoded]) in the resistant Tugela-Dn and a decrease in the synthesis of a 45 kD polypeptide in both resistant Tugela-Dn and susceptible Tugela. Available evidence, molecular mass and high content suggest that the 56 kD protein is Rubisco (Van der Westhuizen and Botha, 1993). Studying susceptible Tugela and the near-isogenic resistant cultivar Tugela-DN, Van Der Westhuizen and Pretorius (1995) concluded that changes in the chlorophyll, protein, free amino acid, proline levels and respiration rate in response to D. noxia infestation indicate that a stress condition is induced in both susceptible and resistant wheat plants by D. noxia feeding. The unique changes in resistant wheat, especially the marked increase in the total free proline content, seems to contribute to the plants improved ability to cope with D. noxia infestation and therefore survive. Proline is known to play a protective role for membrane systems under stress; thus, membranes in resistant plants remain intact and photosynthesis can proceed relatively normally as opposed to susceptible plants where the chloroplasts are damaged. Although an increase in the total phenolic content in infested resistant plants may contribute a possible deterrent effect against D. noxia, none of the other observed biochemical changes in resistant wheat could be regarded as detrimental to D. noxia.

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TABLE 1.1: List of studies conducted with D. noxia resistant donor accessions

Accession and Topic of Study Reference PI 137739

Components of resistance Du Toit (1989b)

Population development and plant damage Quisenberry and Schotzko (1994) Reproductive rate and population development Rafi, Zemetra and Quisenberry (1996)

Feeding damage Rafi, Zemetra and Quisenberry (1997)

DIMBOA concentration Ni and Quisenberry (2000)

PI 140207

Reproductive rate and population development Rafi et al. (1996)

PI 262660

Population development and plant damage Quisenberry and Schotzko (1994) Reproductive rate and population development Rafi et al. (1996)

PI 294994

PI 372129

Winterkill, osmotic potential and fructan content Storlie et al. (1993)

Furthermore the resistance in TugelaDn is associated with elicitor-active, intercellular, infestation-related glycoproteins in the 28-33 kDa range (Van der Westhuizen and Pretorius, 1996). D. noxia

infestation dramatically changed intercellular protein composition of resistant wheat with differential induction of -1,3-glucanase (Van der Westhuizen et al., 1998a), chitinase and peroxidase (Van der Westhuizen et al., 1998b) while in the absence of D. noxia the apoplastic fluid of resistant and susceptible near-isolines was similar. These enzymes, also known as PR-proteins, were induced systemically and are known to be associated with plant defence against invading pathogens. More detailed studies of -1,3-glucanase in planta using an immunogold labelling technique (Van der Westhuizen et al., 2002) showed that -1,3-glucanase accumulated in tissues of resistant wheat most affected by aphid feeding, in particular the cell walls of vascular bundle cells and the chloroplasts. PR-protein activity in resistant wheat has also been shown by Ni et al. (2001) who reported that D. noxia feeding elicited a moderate increase (approximately threefold) of peroxidase specific activity in Halt which contributed to the resistance of this cultivar.

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TABLE 1.2: List of studies conducted with D. noxia resistant cultivars TugelaDn and Halt

Cultivar and Topic of Study Reference TugelaDn

Composition and synthesis of water soluble proteins Van der Westhuizen and Botha (1993) Free proline, total phenolic content and respiration rate Van der Westhuizen and Pretorius (1995) Protien composition of apoplastic fluid Van der Westhuizen and Pretorius (1996) Apoplastic peroxidase and chitinase activities Van der Westhuizen, Qian and Botha (1998b) ß-1-3-glucanase activity Van der Westhuizen, Qian and Botha (1998a) Expression of chitinase isoenzymes Botha, Nagel, van der Westhuizen and Botha (1998) Purification and localisation of ß-1-3-glucanase induced

by D. noxia feeding Van der Westhuizen, Qian, Wilding and Botha (2002)

Salicylic acid in the resistance response of wheat to D.

noxia Mohase and van der Westhuizen (2002)

Trichome presence and leaf epiculticular wax

ultrastructure Bahlmann et al. (2003)

Enzymatic chlorophyll degradation Wang, Quisenberry, Ni and Tolmay (2004b) Photosynthetic pigment concentrations and chlorphyll /

carotenoid ratios Wang, Quisenberry, Ni and Tolmay (2004a)

Halt

Distribution of D. noxia salivary sheaths Ni and Quisenberry (1997a)

Leaf epicuticular structure Ni and Quisenberry (1997b)

Influence of epicuticular wax on probing and

nymphoposition Ni, Quisenberry, Siegfried and Lee (1998)

Phloem composition Telang, Sandström, Dyerson and Moran (1999)

Oxidative response to D. noxia feeding Ni, Quisenberry, Heng-Moss, Markwell, Sarath, Klucas and Baxendale (2001)

Plant damage and yield response Randolph, Peairs, Kroening, Armstrong, Hammon, Walker and Quick (2003)

Categories of resistance at different growth stages Hawley, Peairs and Randolph (2003) Possible roles esterase, glutathione S transferase and

superoxide dismutase (detoxification enzymes) Ni and Quisenberry (2003) Differential colonisation by two biotypes Qureshi et al. (2005) Yield response and categories of resistance Randolph et al. (2005)

Hydrogen peroxide has been shown to signal the induction of downstream defence reactions in TugelaDn, with salicylic acid acting as a later signal for systemic acquired resistance (Mohase and Van der Westhuizen, 2002). Additionally increased levels of salicylic acid inhibit catalase activity,

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which in turn leads to elevated levels of hydrogen peroxide and consequent amplification of the resistance response. The rapid induction of reactive oxygen species (ROS) such as hydrogen peroxide is known as oxidative burst and has been described for plant resistance to both pathogens and herbivores. The balance between production and metabolism of ROS is important to prevent damage to cells. Moloi (2002) as cited by Van der Westhuizen, 2005 reported the induction of ROS scavenging enzymes with anti-oxidative action shortly after the induction of ROS generating enzymes, confirming the signalling role in D. noxia resistance. Lipoxygenase activity was also found to be selectively induced in infested TugelaDn wheat and is also thought to act as signal molecule in activating defence reactions through the 9-HPOD pathway (Van der Westhuizen, 2005). The observed biochemical responses as described above appear to form part of a combined defence mechanism closely resembling plant defence responses to pathogens.

Hydroxamic acids are present in cereals as ß-glucosides which are enzymatically converted to the corresponding aglycons when plant tissue is damaged (Virtanen and Hietala as cited by Mayoral, Tjalingii and Castañera (1996)). Main aglycones found in cereals are DIMBOA and DIBOA. These compounds are known to confer resistance to a wide range of natural enemies of plants including chewing and sap sucking insects as well as bacterial and fungal diseases (Gianoli and Niemeyer, 1998). A negative correlation was found between D. noxia population and DIBOA content of Hordeum seedlings. Nicol, Copaja, Wratten and Niemeyer (1992) screened worldwide wheat cultivars for hydroxamic acid levels finding that susceptible Betta had a DIMBOA level of 1.29 mmol.kg-1 fresh weight while that of susceptible Tugela was 2.00 mmol.kg-1 and that of the resistant donor accession SA 2199 [PI 262660] 2.15 mmol.kg-1_{all of which fall in the moderate}

level as defined by Givovich and Niemeyer (1996).

The study of aphid-plant interactions using EPG

Prado (1997) defines aphid-plant interaction as comprising of host plant attraction, plant penetration, sap feeding by the aphid and the reactions to these activities by the plant. This is an extremely complex process that has been studied extensively for various aphid host-plant combinations but is not yet entirely understood (Caillaud and Niemeyer, 1996).

Study of the feeding behaviour of piercing-sucking insects like homopterans, is difficult because once the insect inserts its stylets into the plant tissue, relevant behaviours occur within the opaque food substrate and are not directly observable (Walker, 2000). Homopteran probing can however be effectively studied using the electrical penetration graph (EPG) technique (McLean and Kinsey, 1964; Tjallingii, 1978, 1985a, 1985b, 1988) and this method is increasingly being used to study aphid–plant interactions.

Initial attempts at electronic monitoring of insect probing were made in the early 1960’s (McLean and Kinsey, 1964) and with time the systems have been further developed and refined. In principle

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this technique works by connecting the aphid and the plant substrate into an electric circuit, which is completed when the aphid stylet penetrates the plant to feed. A thin gold wire (8-20 µm in diameter) is glued to the insect’s dorsum using water based conductive silver glue and Tjallingii (1986) showed that this does not influence feeding behaviour significantly if correctly attached. A second electrode is connected to the plant or plant substrate. A small voltage (either AC or DC depending on the system used) is applied across the insect and the substrate. Completion of the circuit occurs when aphid stylets penetrate the plant, the current flows and a signal can be recorded. As the electrical impedance fluctuates in the insect-substrate circuit, these impedance fluctuations are superimposed on the rate of charge flow or current in the circuit. By converting the current fluctuations to voltage fluctuations and amplifying the voltage level the impedance changes become signals that can be observed and recorded using electronic devices. The DC system records two signal components originating from the insect-plant interaction namely the resistance or conductivity component (R) and the electromotive forces (emf) actively generated by the insect-plant combination, while the AC system records only the fluctuating voltage over time caused by changes in the electrical resistance of the insect-plant combination (R). Certain repetitive or periodic impedance changes have been correlated with specific behaviours (probing, salivation and ingestion) and with the penetration of certain plant tissue (Kimsey and McLean, 1987) and as systems have been improved and fine-tuned new waveforms and details could be correlated with previously unknown probing activities (Tjallingii, 2000). Some confusion can exist due to existence of both AC and DC systems each with their own peculiarity. The respective equivalents for the AC and DC waveforms are as follows: Salivation (S) for pathway phase (ABC); phloem ingestion (PI) or committed phloem ingestion (CPI) for phloem phase (E); non-phloem ingestion (NPI) for xylem phase.

The EPG technique has applications in the study of virus transmission (Woodford and Mann, 1992; Harrewijn, de Kogel and Piron, 1998), the influence of water deficit (Al-Dawood, Radcliffe, Backus and Koukkari, 1996), the effect of anti-feedant compounds and mineral oils (Powell, Hardie and Pickett, 1998), insecticides [Pymetrozine (Harrewijn and Kayser, 1997), Imidacloprid (Woodford and Mann, 1992; Epperlein and Jaschewski, 1997)] as well as the clarification of the insect-host plant interaction. EPG’s provide the opportunity of localising the resistance mechanism in the plant, be it mechanical or chemical properties of plant tissues (phloem, cuticle, epidermis, mesophyll) (Van Helden and Tjallingii, 2000) thus facilitating the use of pest resistance in crops, an environmentally responsible strategy which is increasingly being deployed for the control of agriculturally significant pests (Van Helden and Tjallingii, 2000; Walker, 2000). EPG’s have been used to study host plant resistance to many hemipterous pests amongst others spotted alfalfa aphid, Therioaphis maculata, on alfalfa (Nielson and Don, 1974); Melon aphid, Aphis gossypii, on muskmelon (Kennedy, McLean and Kinsey, 1978; Klinger, Powell, Thompson and Isaacs, 1998); brown planthopper, Nilaparvata lugens, on rice (Velusamy and Heinrichs, 1986); leafhopper,

Nephotettix virescens, on rice (Rapusas and Heinrichs, 1990); black cowpea aphid, Aphis cracivora, on cowpea (Mesfin Thottapilly and Singh, 1992); Greenbug, Schizaphis graminum, on wheat (Morgham, Richardson, Campbell and Eikenberry, 1992); cabbage aphid, Brevicoryne